Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs

Article abstract of DOI:10.3109/1061186X.2010.529909

The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enz

Full text of DOI:10.3109/1061186X.2010.529909

Journal of Drug Targeting, 2010; 18(10): 794–802
© 2010 Informa UK, Ltd.
ISSN 1061-186X print/ISSN 1029-2330 online
DOI: 10.3109/1061186X.2010.529909
ORIGINAL ARTICLE
Lectin-directed enzyme activated prodrug therapy (LEAPT):
Synthesis and evaluation of rhamnose-capped prodrugs
Philippe Garnier1, Xiang-Tao Wang1, Mark A. Robinson1, Sander van Kasteren1, Alan C. Perkins2,
Malcolm Frier2, Antony J. Fairbanks1,3, and Benjamin G. Davis1
1Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK, 2Department of Medical
Physics, Medical School, Queen’s Medical Centre, Nottingham, UK, and 3Department of Chemistry, University of
Canterbury, Christchurch, New Zealand
Abstract
The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated
enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the
sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and
5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated
for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase
enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with
their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that
may be used in LEAPT.
Keywords: Lectins, carbohydrates, doxorubicin, 5-fluorouracil, prodrug, enzyme release, l-rhamnose, d-galactose
Introduction
on non-cancerous ones, has been cleverly addressed
by using bipartite systems such as antibody-directed
enzyme prodrug therapy, using monoclonal antibody
(mAb)–enzyme conjugates to release prodrugs at pre-
determined sites (Bagshawe, 1994). Similarly, catalytic
antibodies have been suggested in a potential antibody-
directed abzyme prodrug therapy (Wentworth et al.,
1996). Both methods rely on the localization of the bio-
catalytic entity—an enzyme or a catalytic antibody—at
a site determined by specific interactions with antigens
presented on cells; a drug is subsequently released from
its prodrug by this localized enzyme.
An approach that could exploit specific endogenous
localization interactions could present clear advan-
tages with regard to selectivity, mechanism, and avoid-
ing the need to induce an effective, tolerated mAb as
a part of a subsequent construct. We have previously
described a novel catalytic, bipartite drug delivery sys-
tem, lectin-directed enzyme activated prodrug therapy
Carbohydrate-based ligand–receptor mechanisms are
involved in a number of key cellular processes such as
immune response (Varki, 1993), cell surface communica-
tion and signaling (Dwek, 1996), as well as inflammation
(Weis and Drickamer, 1996; Zhang et al., 2010). Given the
high specificity of interaction with carbohydrates and the
broad range of cellular receptors that can potentially be
specifically targeted by glycosylated molecules, a number
of glycoprotein and glycopolymer-based systems have
been developed to deliver drugs selectively to the desired
active sites (Davis and Robinson, 2002; Singh et al.,
2008). However, many of these systems are plagued by
unwanted drug release at sites different from the desired
site of action due to the very nature of the release mecha-
nisms involved, for example endogenous lysosomal deg-
radation. e challenge of achieving high site selectivity,
which is for example of particular importance for the
treatment of cancer cells while avoiding cytotoxic effects
e first two authors contributed equally to this work.
Address for Correspondence: Benjamin G. Davis, Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield
Road, Oxford OX1 3TA, UK. E-mail: ben.davis@chem.ox.ac.uk
(Received 11 August 2010; revised 03 October 2010; accepted 04 October 2010)
794

Synthesis and evaluation of rhamnose-capped prodrugs 795
(LEAPT), which exploits the selectivity and specificity
of carbohydrate–protein interactions to localize glyco-
conjugates in a switchable and sugar-dependent manner
(Robinson et al., 2004). In the first phase of this strategy
a glycosylated enzyme is targeted to specific cell types
or tissues (Scheme 1). In the second phase, prodrugs
capped with sugars are then administered. e glyco-
sylated enzyme is then able to activate the prodrugs at
the site of interest by cleaving the prodrug linkage. e
colocalization of both prodrug and enzyme relies on
their precise glycosylation. Selective enzyme targeting
can be achieved by modification of the enzyme with car-
bohydrates that bind specific lectins on the surface of the
targeted cells. Asialoglycoprotein receptors (ASGPRs)
are expressed in abundance on the surface of hepato-
cytes (Lis and Sharon, 1998) and can specifically bind the
sugars d-galactose (d-Gal) and l-rhamnose. Moreover,
ASGPRs have the ability to trigger receptor-mediated
endocytosis (RME) and transfer their ligands inside the
cell. erefore, we designed a system in which drugs of
interest are capped with rhamnose and are released by
a rhamnosidase enzyme, which is by itself gets glycosy-
lated with Gal. For these potential prodrugs, the rham-
noside cap would serve (1) to decrease the cytotoxicity
of the drug before its release, (2) to deliver the prodrug
to specific cell types by RME—in this case hepatocytes
after binding to ASGPR and subsequent endocytosis,
and (3) to increase the sometimes poor solubility of
drugs used in certain therapies. Galactosylation of the
rhamnosidase would enable ASGPR-mediated internal-
ization of this enzyme into the hepatocytes by RME. e
non-mammalian sugar l-rhamnose (Rha)—and its asso-
ciated glycosidase (l-rhamnosidase)—was chosen as a
sugar cap to avoid prodrug activation at unwanted sites
by endogenous mammalian glycosidases before reach-
ing the ASGPR.
of malignancies such as breast cancer, Hodgkin’s and
non-Hodgkin’s lymphoma, and acute leukemia (Blum
and Carter, 1974; Launchbury and Habboubi, 1993).
However, Dox is plagued by adverse effects and its car-
diotoxicity, in particular, is well established (Lefrak et al.,
1973), and like other anthracyclins, it is associated with
cardiomyopathies that lead to congestive heart failures.
5Fu is an inhibitor of thymidilate synthase and is a major
anticancer agent used in the treatment of gastrointesti-
nal malignancies and breast cancer, and its combination
with other drugs has had a great impact on the treatment
of colorectal cancer (Longley et al., 2003), despite few
low-response rates, for example in the first-line treat-
ment of advanced colorectal cancer (Johnston and Kaye,
2001). e stability of the two prodrugs in blood was then
evaluated to assess whether they are stable to circulating
glycosidases. As the vital second component, rhamnosi-
dase was modified with galactose (Gal) residues, and
the biodistribution of this glycoengineered enzyme was
determined in vivo by gamma scintigraphy.
Materials and methods
Synthesis—general
Proton nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker DQX 400 (400 MHz) spectrometer,
andthespectrawereassignedusingCOSY.Carbonnuclear
magnetic resonance spectra were recorded on a Bruker
DQX 400 (100.6 MHz) spectrometer and were assigned
using HMQC. Multiplicities were assigned using DEPT
sequence. All chemical shifts are quoted on the δ scale
in parts per million using residual solvent as the internal
standard. Low-resolution mass spectra were recorded on
a Micromass Platform 1 spectrometer using electrospray
ionisation (ESI). High-resolution mass spectra were
recorded by Dr. Neil J. Oldham in the Chemistry Research
Laboratory on a Walters 2790 Micromass LCT ESI mass
spectrometer using chemical ionization (NH3, Cl) tech-
niques as stated; m/z values are reported in dalton and are
followed by their percentage abundance in parentheses.
Infra red spectra were obtained with a Bruker Tensor 27
spectrophotometer, adsorption maxima being recorded
in wave numbers (cm−1) and classified as s (strong) and
br (broad). in layer chromatography (TLC) was carried
To validate this strategy and assess its therapeutic
potential, we set out to prepare representative rhamnose-
capped prodrugs, Rha-doxorubicin (Dox) and Rha-5-
fluorouracil (5Fu), the two known cytotoxic compounds
(Scheme 2).
Anthracycline doxorubicin, which intercalates in
DNA and inhibits topoisomerase activity, has been
used since the 1960s in the treatment of a wide range
Rhamnosidase
O
X
enzyme
1
O
O
X
X
RME
Sugar
targeting
ligand
3
Drug
Drug
Drug
Drug
Rha-capped
prodrug
O
RME
O
O
O
O
O
HO
HO
HO
HO
OH
L-rhamnose
(Rha)
HO
OH
HO
2
OH
Scheme 1. Concept of the LEAPT strategy (Robinson et al., 2004).
© 2010 Informa UK, Ltd.

796 P. Garnier et al.
2.0 Hz, H-1) ppm; 13C NMR (100MHz, CDCl3): δ=17.2
(C-6), 20.7, 20.7, 20.8, 20.9 (4×COCH3), 68.7, 70.4 (C-2,
C-5), 90.6 (C-1), 168.4, 169.8, 169.8, 170.1 (4×4×COCH3)
ppm; m/z (ES+) 355 (M–Na, 100%).
out on Merck TLC silica gel 60 F254 aluminium plates.
Visualization of the plates was achieved using a UV lamp
(λmax = 254 or 365 nm), and/or ammonium molybdate (5%
in 2 M H2SO4) or sulfuric acid (5% in EtOH). Flash col-
umn chromatography was carried out using Sorbsil C60
40/60 silica. DCM was distilled from calcium hydride.
THF was distilled from sodium wire and benzophenone.
Remaining anhydrous solvents were purchased from
Fluka and Acros. “Petrol” refers to the fraction of petro-
leum ether boiling in the range 40–60°C.
2,3,4-Tri-O-acetyl-l-rhamnose (3)
1,2,3,4-Tetra-O-acetyl-l-rhamnose
2
(20.15g, 60.7
mmol) was dissolved in dry THF (160ml) under argon.
Benzylamine (10ml, 91.5 mmol) was added and the mix-
ture was stirred at room temperature for 18h before addi-
tion of water (300ml). After extraction with DCM (450ml,
then 2×150ml), the combined organic fractions were
washedwith10%HCl(100ml),saturatedNaHCO3 (100ml),
and brine (100ml), dried over MgSO4, and concentrated
in vacuo. Purification by flash column chromatography
(AcOEt/petrol 2/3) afforded 2,3,4-tri-O-acetyl-l-rham-
nose 3 (16.0g, 91%) as a white powder and a mixture of α-
and β-anomers. For the major anomer α: Rf 0.38 (AcOEt/
petrol 1/1); 1H NMR (400MHz, CDCl3): δ=1.22 (3H, d, J5,6
6.3 Hz, H-6), 2.00, 2.06, 2.16 (3×3H, 3×s, 3×COCH3), 4.95
(0.2 H, bs, OH), 4.10–4.16 (1H, m, H-5), 5.08 (1H, t, J4,5 9.9
Hz, H-4), 5.15–5.17 (1H, m, H-1), 5.27 (1H, dd, J1,2 1.8 Hz, J2,3
3.3 Hz, H-2), 5.37 (1H, dd, J2,3 3.3 Hz, J3,4 9.9 Hz, H-3) ppm;
13C NMR (100MHz, CDCl3): δ=17.4 (C-6), 20.7, 20.8, 20.9
(3×COCH3), 66.4 (C-5), 68.8 (C-3), 70.2 (C-2), 71.1 (C-4),
92.1 (C-1), 170.1, 170.2, 170.3 (3×COCH3) ppm.
Prodrug synthesis
1,2,3,4-Tetra-O-acetyl-l-rhamnose (1)
l-Rhamnosemonohydrate(12g,0.66mmol)wasdissolved
in acetic anhydride (40ml) and pyridine (40ml) and the
mixture was stirred overnight at room temperature. e
reaction mixture was diluted with AcOEt (300ml), poured
into ice water (500ml), and stirred for 1h. e aqueous
fraction was separated and extracted twice with AcOEt
(2×250ml). e combined organic fractions were washed
with 5% HCl (100ml), saturated NaHCO3 (2×100ml),
water (100ml), and brine (100ml); dried over MgSO4;
and concentrated in vacuo. Purification by flash column
chromatography (AcOEt/petrol 1/2) afforded 1,2,3,4-tet-
ra-O-acetyl-l-rhamnose 2 (22.5g, quantitative) as a color-
less syrup and a mixture of α- and β-anomers (α/β 1/3).
For the α-anomer: Rf 0.49 (AcOEt/petrol 1/1); 1H NMR
(400MHz, CDCl3): δ=1.23 (3H, d, J5,6 6.3 Hz, H-6), 2.00,
2.06, 2.16, 2.21 (4×3H, 4×s, 4×COCH3), 3.90–3.97 (1H, m,
H-5), 5.12 (1H, at, J=9.9 Hz, H-4), 5.24–5.25 (1H, m, H-2),
5.30 (1H, dd, J2,3 3.2 Hz, J3,4 9.9 Hz, H-3), 6.01 (1H, d, J1,2
(2,3,4-Tri-O-acetyl-α-l-rhamnopyranosyl) 4-nitrophenyl
carbonate (4)
2,3,4-Tri-O-acetyl rhamnose 3 (15.90 g, 54.4 mmol) and
4-nitrophenyl chloroformate (11.6 g, 57.5 mmol) were
O
O
OH
O
O
O
OH
O
O
O
OH
OH
O
OH
OH
OH
OH
OH
OH
CH3O
OH
O
CH3O
OH
O
CH3O
O
rhamnosidase
O
O
O
NH
HO
NH
NH2
HO
HO
O
O
doxorubicin(Dox)
-O
O
O
HO
HO
OH
Rha-Dox
O
O
F
−O
N
N
N
F
rhamnosidase
H
N
O
N
H
O
N
H
O
O
O
N
H
O
HO
F
HN
HO
OH
Rha-5Fu
F
H2N
O
N
H
O
O
N
H
O
5-fluorouracil(5Fu)
Scheme 2. Doxorubicin and 5-fluorouracil prodrugs and their postulated activation mechanisms by rhamnosidase.
Journal of Drug Targeting

Synthesis and evaluation of rhamnose-capped prodrugs 797
dissolved in dry DCM (160 ml) under an atmosphere of
argon. Pyridine (4.4 ml, 54.4 mmol) was added, and the
mixture was stirred overnight at room temperature. e
reaction mixture was concentrated in vacuo. e residue
was purified by flash chromatography (AcOEt/petrol 2/3)
to afford compound 4 (14.87 g, 60%) as an yellow powder.
Rf 0.4, AcOEt/petrol 2/3); [α]21D = −67.7 (c 1, CHCl3);νmax
(KBr) 1790, 1753 (s, CO), 1619 (s, Ar), 1533 (s, NO2), 1351
(s, NO2) cm−1; 1H NMR (400 MHz, CDCl3): δ= 1.30 (3H, d,
3 × s, 3 × COCH3), 3.89–3.96 (1H, m, H-5), 5.11 (1H, dd,
J3,4 10.1 Hz, J4,5 9.9 Hz, H-4), 5.19-5.25 (1H, m, H-3), 5.22
(2H, s, H8), 5.27 (1H, dd, J1,2 1.5 Hz, J2,3 3.3 Hz, H-2), 6.00
(1H, d, J1,2 1.5 Hz, H-1), 6.10 (1H, pt, JNH–CH2 7.1 Hz, NH)
ppm; 13C NMR (100 MHz, CDCl3): δ 17.4 (C-6), 20.6, 20.8,
20.8, 20.9 (4 × COCH3), 66.2 (CH2), 68.5, 68.6, 68.7 (C-2,
C-3, C-5), 70.3 (C-4), 91.8 (C-1), 153.1 (OC(O)N), 169.8,
169.8, 170.1 (3 × COCH3), 171.8 (COCH3) ppm; m/z (ES+)
423 (M + NH4 , 30%), 428 (MNa+, 100%); HRMS (ES+)
+
J5,6 6.1 Hz, H-6), 2.04, 2.09, 2.20 (3 × 3H, 3 × s, 3 × COCH3),
calculated for C16H23NO11Na (MNa+) 428.1169, found
428.1165.
4.04–4.16 (1H, m, H-5), 5.19 (1H, at, J 10.1 Hz, H-4), 5.37
(1H, dd, J2,3 3.5 Hz, J3,4 10.1 Hz, H-3), 5.43 (1H, dd, J1,2 2.0
Hz, J2,3 3.5 Hz, H-2), 6.02 (1H, d, J1,2 1.8 Hz, H-1), 7.46 (2H,
d, J 9.1 Hz, Ar-Ha), 8.31 (2H, d, J 9.1 Hz, Ar-Hb) ppm; 13C
NMR (100 MHz, CDCl3): δ= 17.4 (C-6), 20.6, 20.7, 20.7
(3 × COCH3), 68.2 (C-2), 68.4 (C-3), 69.4 (C-5), 70.0 (C-4),
95.2 (C-1), 121.7 (Ara), 125.4 (Arb), 145.7 (CNO2), 150.4,
155.0 (Ar-O, OC(O)O), 169.7, 169.8, 170.0 (COCH3) ppm.
N-(2,3,4-tri-O-acetyl-α-l-rhamnopyranosyloxycarbonyl)-
(5-fluoro-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-
methylamine (7)
Compound 6 (169 mg, 0.417 mmol) was dissolved in
dry DMF (10 ml). Triethylamine (60 µl, 0.43 mmol)
and 5Fu (178 mg, 1.36 mmol) were successively added
and the reaction mixture was stirred at room tempera-
ture for 16 h. e reaction mixture was concentrated in
vacuo. e white residue was dissolved in water (50 ml)
and AcOEt (50 ml). e organic fraction was separated
and the aqueous fraction was extracted with AcOEt
(2 × 50 ml). e combined organic fractions were washed
with brine (20 ml), dried over MgSO4, and concentrated
in vacuo. Purification by flash column chromatog-
raphy (AcOEt) afforded compound 7 (154 mg, 77%).
[α]21D = −60.0 (c 1, CHCl3); νmax (KBr) 3410 (b, NH), 1751
N-(2,3,4-Tri-O-acetyl-α-l-rhamnopyranosyloxycarbonyl)
glycine (5)
Compound 4 (2.23 g, 7.10 mmol) was dissolved in 75 ml
of acetone. Water (40 ml), NaHCO3 (1.81 g, 21.5 mmol)
and glycine (1.62 g, 21.6 mmol) were added. e reac-
tion mixture was stirred for 3 h at room temperature
until no starting material could be detected by TLC. e
reaction mixture was neutralized with 20% HCl to pH 3.
After extraction with AcOEt (3 × 100 ml), the organic frac-
tions were combined, washed with brine (100 ml), dried
over MgSO4, and concentrated in vacuo. Purification
by flash column chromatography (AcOEt, 1% MeOH)
afforded compound 5 as a white powder (1.54 g, 80%).
1
(s, CO), 1234 (s, CF) cm−1; H NMR (400 MHz, CDCl3):
δ= 1.23 (3H, d, J5,6 6.1 Hz, H-6), 1.99, 2.05, 2.16 (3 × 3H,
3 × s, 3 × COCH3), 3.94–4.01 (1H, m, H-5), 4.99–5.14 (3H,
m, H-8, H-4), 5.23–5.28 (2H, m, H-3, H-2), 5.96 (1H, bs,
H-1), 7.35 (1H, t, JNH–CH2 6.6 Hz, NHCH2), 7.71 (1H, d, J5,6
1
[α]21D = −34.6 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3):
δ= 1.24 (3H, d, J5,6 6.1 Hz, H-6), 2.03, 2.07, 2.18 (3 × 3H,
3 × s, 3 × COCH3), 3.97 (1H, dd, J4,5 9.9 Hz, H-5), 4.08–4.14
(1H, m, CH2), 5.13 (1H, pt, J3,4 10.1 Hz, H-4), 5.28–5.30
(1H, m, H-2), 5.34 (1H, dd, J1,2 2.0 Hz, H-3), 5.76 (1H, t,
JNH–CH2 5.3 Hz, NH), 5.96 (1H, d, J1,2 2.0 Hz, H-1) ppm;
13C NMR (100 MHz, CDCl3): δ 17.4 (C-6), 20.7, 20.8, 21.1
(3 × COCH3), 42.4 (CH2), 68.4, 68.7, 68.9 (C-2, C-3, C-5),
70.5 (C-4), 91.6 (C-1), 153.6 (OC(O)N), 169.9, 170.0, 170.8
(3 × COCH3), 173.0 (CO2H) ppm; m/z (ES−) 390 (M–H,
100%); HRMS (ES−) calculated for C15H20NO11 (M-–H)
390.1036, found value 390.1055.
5.1 Hz, H-6'), 10.25 (1H, d, J 4.5 Hz, CONHCO) ppm; 13
C
NMR (100 MHz, CDCl3): δ= 17.4 (C-6), 20.7, 20.7, 20.8
(3 × COCH3), 55.2 (C-8), 68.6, 68.6, 68.7 (C-2, C-3, C-5),
70.3 (C-4), 91.9 (C-1), 129.3 (C-6'), 141.4 (C-5'), 150.2
(C-2'), 154.6 (OC(O)N), 157.5 (C-4'), 169.8, 169.9, 170.3
(3 × COCH3) ppm; m/z (ES−) 474 (M–H+, 100%); HRMS
(ES−) calculated for C18H21N3O11F (M–H+) 474.1160, found
474.1181.
N-(α-l-rhamnopyranosyloxycarbonyl)-(5-fluoro-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-1-yl)-methylamine (8)
Compound 7 (124mg, 0.26 mmol) was dissolved in dry
MeOH (6ml) under argon. e solution was cooled
to 0°C before addition of sodium methoxide (22mg,
0.42 mmol). After 1h, the reaction was quenched by addi-
tion of Dowex-H+. e mixture was filtered, concentrated,
and purified by flash chromatography (AcOEt/MeOH
5/1) to afford compound 8 (91mg, 99%) as a white pow-
der. [α]21D =−27.4 (c 0.5, MeOH); νmax (KBr) 3415 (b,OH,
Acetoxy-N-(2,3,4-tri-O-acetylα-l-
rhamnopyranosyloxycarbonyl) methylamine (6)
Compound 5 (500 mg, 1.28 mmol) was dissolved in a
mixture of dry THF (30 ml) and toluene (6 ml) under
argon. Pyridine (105 µl, 1.30 mmol) and lead tetra-ac-
etate (715 mg, 1.61 mmol) were successively added and
the mixture was refluxed (70°C) for 45 min. e reaction
was cooled to room temperature and filtered over Celite®.
e filtrate was concentrated in vacuo. e residue was
purified by flash chromatography (AcOEt/petrol 1/1) to
afford compound 6 (185 mg, 35%) as a white powder.
1
NH), 1699, 1654 (s, CO, C=C), 1243 (s, CF) cm−1; H NMR
(400MHz, CD3OD): δ=1.26(3H, d, J5,6 6.1 Hz, H-6), 3.42 (1H,
pt, J 9.6 Hz, H-4), 3.60–3.70 (2H, m, H-3, H-5), 3.73–3.84 (1H,
m, H-2), 5.02 (2H, s, H-8), 5.88 (1H, d, H-1) ppm; m/z (ES−)
348 (MH+, 30%), 384 (MCl−, 100%); HRMS (ES−) calculated
for C12H15N3O8F (M–H+) 348.0843, found 348.0836.
1
[α]21D = −28.6 (c 0.3, CHCl3); H NMR (400 MHz, CDCl3):
δ= 1.25 (3H, d, J5,6 6.3 Hz, H-6), 2.00, 2.11, 2.17 (3 × 3H,
© 2010 Informa UK, Ltd.

798 P. Garnier et al.
N-(2,3,4-tri-O-acetyl-α-l-rhamnopyranosyloxycarbonyl)
doxorubicin (10)
Preparation of deglycosylated α-l-rhamnosidase
(N-DG)
Compound 4 (1.57 g, 3.44 mmol) and doxorubicin
hydrochloride 9 (2.00 g, 3.44 mmol) were dissolved in
dry DMF (125 ml). Triethylamine (0.7 ml, 5.2 mmol)
was added and the mixture was stirred overnight before
being concentrated in vacuo. e residue was purified
by flash chromatography (AcOEt, then AcOEt/MeOH
9/1) to afford compound 10 (2.77 g, 94%) as a red powder
(Rf 0.18, AcOEt). [α]21D = +12.6 (c 1, CHCl3); m/z (ES−) 858
(M–H, 50%); HRMS (ES+) calculated for C40H49N2O20 (M +
NH4) 877.2879, found 877.2874.
Wild-type naringinase (N-WT) was deglycosylated using
endoglycosidase-H (endo-H, 32 U/100 mg N-WT) for 6 h
in 0.1 M orthophosphate buffer, pH 6.0, 37°C, and then
purified by dialysis (50 kDa MWCO) to give deglycosy-
lated naringinase (N-DG; Robinson et al., 2004).
Protein glycosylation to yield N-DG-Gal and N-WT-Gal
Proteins were glycosylated using the 2-imino-2-
methoxyethyl (IME) 1-thioglycoside method (Stowell and
Lee, 1980, 1982). An approximately 500:1 ratio of modifi-
cation reagent to protein was used in each reaction. For
example,N-DG-Galwaspreparedasfollows.Cyanomethyl-
2,3,4,6-tetra-O-acetyl-β-d-thiogalactopyranoside (30mg,
0.074 mmol) was dissolved in anhydrous methanol
(3.0ml) in a flask fitted with a magnetic stirrer under an
inert atmosphere, treated with an anhydrous methanolic
solution of sodium methoxide (30 μl, 1M) and stirring
continued at room temperature. After 36h, all solvent was
removed in vacuo yielding a white gum. N-DG (10mg)
was dissolved in an aqueous solution 0.25M sodium tet-
raborate (2ml) pH 8.5, added to the white gum, and stirred
at room temperature for 24h. e solution was dialyzed
against deionised water (2 l, 4 changes) using Visking dial-
ysis tubing (12-14kDa MWCO), and then desalted using
Sephadex G25 (PD10 column), eluting with deionised
water. e resultant solution was freeze-dried yielding a
white powder (Robinson et al., 2004).
N-α-l-rhamnopyranosyloxycarbonyl) doxorubicin (11)
Compound 10 (320 mg, 0.37 mmol) was dissolved in a
mixture of 50 ml dry MeOH, 10 ml dry DMF, and 2.5 ml
dry THF. e solution was cooled to 0°C before NaOMe
(54 mg, 1 mmol) dissolved in 1 ml dry MeOH was added.
ereactionwascarefullyfollowedbyTLC,andquenched
after 1 h and 40 min with Dowex-H+ when no starting
material could be detected. e mixture was filtered and
concentrated in vacuo to afford compound 11 (265 mg,
94%) as a red powder. [α]21D = +19.6 (c 0.15, MeOH); ν IR
1
(KBr) ν (CHCl3), 4300 (OH, NH), 1725 cm−1 (NHCO); H
NMR (400 MHz, H2O): δ= 1.19 (3H, d, H-6'), 1.32 (3H, d,
H-6″), 1.78–192 (3H, m, H-2', H-8a), 2.11 (1H, d, H-8b),
2.39 (1H, d, H-10a), 2.70 (1H, d, H-10b), 3.60–3.72 (7H, m,
H-3', H-3″, H-5″, H-4″,OCH3), 3.73–3.89 (1H, m, H-2″),
4.08–4.14 (1H, m, H-5'), 4.55 (1H, bs, H-7), 5.29 (1H, bs,
H-1'), 5.88 (1H, d, H-1″), 7.1 (1H d H-1), 7.10 (1H, d, H-1),
7.35 (1H, t, H-2) ppm; m/z (ES−) 732 (M–H, 100%); HRMS
(ES−) calculated for C34H38NO17 (M–H) 732.2140, found
732.2139.
Protein analysis
Proteins were analyzed by gel electrophoresis (10%
SDS–PAGE, pH 8.8, Tris buffer; Vertical Slab Gel Kit,
Atto Corporation), and by matrix-assisted laser des-
orption ionization mass spectrometry (MS, Ciphergen
Biosystems PBS II, Ciphergen Biosystems; sinapinic acid
matrix 10 mg/ml 3:2 water:acetonitrile, 0.2% TFA).
Purification of α-l-rhamnosidase (N-WT)
α-l-Rhamnosidase from Penicillium decumbens
(naringinase, EC 3.2.1.40) is a commercially avail-
able (Sigma–Aldrich) enzyme preparation composed
of α-rhamnosidase and β-glucosidase activities.
Purification and deglycosylation of naringinase was car-
ried out using methods described previously (Robinson
et al., 2004). Naringinase was purified to yield pure wild-
type α-rhamnosidase activity in four steps: (1) dialysis
(12–14 kDa MWCO, Visking dialysis tubing); (2) BioGel
P100 size exclusion chromatography (BioRad, elu-
ant pH dionised water); (3) BioGel P100 size exclusion
chromatography (BioRad, eluant pH 4.8, 0.1 M NaCl);
(4) DEAE Sepharose ion exchange chromatography
(Amersham Biosciences, eluant pH 6.0, 20 mM l-his-
tidine 0–0.35 mM NaCl gradient). In Steps 3 and 4, the
fractions containing solely α-l-rhamnosidase activity
were combined, freeze-dried, and desalted by dialysis.
α-l-Rhamnosidase activity was assessed using para-ni-
trophenyl α-l-rhamnopyranoside (para-NP α-l-Rha) as
substrate by mixing equal volumes of fraction constitu-
ent with 3.5 mM para-NP α-l-Rha in orthophosphate
buffer and determining release of para-nitrophenol at
405 nm.
Prodrug stability study in blood
Rha-Dox (11) solutions (1, 10, or 100 μg/ml) were
prepared in either 0.9% NaCl or rabbit blood and incu-
bated at 37°C. For the samples incubated in 0.9% NaCl,
aliquots (0.5 ml) collected at given time points were mixed
with 20% TFA (15 μl), transferred into a Vivaspin 3000
tubes and centrifuged at 13,000g for 30 min. e filtrate
was directly injected for HPLC determination. For blood
samples, the blood was centrifuged at 13,000g for 10 min,
0.5 ml of supernatant was mixed with 20% TFA (15 μl),
sonicated at 25°C for 5 min, transferred into Vivaspin
3000 tubes, and centrifuged at 13,000g for 30 min. e
filtrate was analyzed by HPLC.
Similarly, Rha-5Fu (8) was incubated in 0.9% NaCl or
rabbit blood at 37°C at 2, 20, or 200 μg/ml. For the sam-
ples incubated in 0.9% NaCl, aliquots (0.2 ml) collected
at given time points were mixed with 20% TFA (12 μl)
and deionised water (188 μl), sonicated at 25°C for 5 min,
and centrifuged at 13,000g for 10 min. e supernatant
was transferred into Vivaspin 3000 tubes, centrifuged at
Journal of Drug Targeting

Synthesis and evaluation of rhamnose-capped prodrugs 799
13,000g for 30 min, and 50 μl of filtrate was used for HPLC
analysis. For blood samples, the blood was centrifuged at
13,000g for 10 min, 0.2 ml of plasma was diluted with TFA
and deionised water, and then sonicated and centrifuged
as described earlier.
Table 1. HPLC conditions for the 5Fu and Rha-5Fu samples.
Mobile
phase
Flow rate Retention
(ml/min) time (min)
Time (min)
0–12
5Fu
MeOH–H2O
(99:1)
0.5
9.8
12–19
19–26
0–15
MeOH–H2O
(97:3)
2
Analyses were performed on a reverse-phase HPLC
system consisting of a Waters 2795 separations module
(Waters Assoc. Milford, MA) coupled to a Jasco FP-920
Intelligent fluorescence detector (Waters Assoc.) for
the Dox analysis (excitation wavelength set at 480 nm
and emission at 560 nm, gain was set at ×100). For the
5Fu analysis, a Waters 2996 photodiode array detector
was used (detection wavelength was set at 265 nm for
5Fu and 269 nm for Rha-5Fu). Chromatograms were
acquired, stored, and processed using the Empower
software program (Waters). Chromatographic separa-
tions were carried out using a Hypersil C8 analytical
column (5 μm, 250 × 4.6 mm). e Dox samples were
separated under isocratic conditions at 65% sodium
phosphate buffer (20 mM, pH 2.8, containing 0.01%
TFA) and 35% methanol at 1.0 ml/min. e mobile
phases used for the 5Fu samples are summarized in
Table 1.
MeOH–H2O
(99:1)
2
Rha-5Fu
MeOH–H2O
(97:3)
1
11.9
monohydrate (1) was peracetylated by reaction with
acetic anhydride and pyridine (Fisher et al., 1920).
Deprotection of the anomeric hydroxyl was achieved
after treatment with benzylamine to afford compound
3 (Sim et al., 1993). Hemiacetal 3 was then reacted with
4-nitrophenyl chloroformate in the presence of pyridine
to afford mixed carbonate 4 in 60% yield. Compound 4
was the key divergent intermediate used for the prepara-
tion of both the prodrugs.
Prodrug 8 was prepared from activated rhamnose
precursor 4 as follows (Scheme 3). Coupling of com-
pound 4 with glycine in DMF, to install the first portion
of the aminal-carbamate linker, was initially unsuccess-
ful. After further attempts at varying reaction conditions
(with triethylamine or N-ethyldiisopropylamine, under
phase transfer conditions, under reflux), glycinylcarbam-
ate 5 was eventually obtained in 80% yield using sodium
bicarbonate as base and a mixture of water and acetone
as solvent. Oxidative decarboxylation (Gledhill et al.,
1986; Madec-Lougerstay et al., 1999) of compound 5
with lead tetra-acetate (1.25 eq.) in the presence of pyri-
dine (1 eq.) was achieved in 35% yield to afford acetate
6 after purification by flash chromatography on silica.
Condensation of compound 6 with 5Fu in the presence
of Et3N proceeded in 77% yield to afford compound
7. We suspected the product to be unstable due to its
hemiaminal-like structure, and indeed 2D-TLC on silica
revealed partial degradation. erefore, subsequently no
attempt was made to purify compound 6 before coupling
with 5Fu; decarboxylation of compound 5 was performed
by heating lead tetra-acetate for 40 min under reflux; the
reaction mixture was then cooled to room temperature
and filtered through Celite®. Immediate coupling of the
crude product with 5Fu yielded acetylated prodrug 7 in
97% yield over two steps. Finally, deacetylation under
standard Zémplen conditions afforded 5Fu prodrug 8 in
quantitative yield.
Preparation of 123I-labeled enzymes
e glycosylated enzymes were labeled with 123I by
using
1,3,4,6-tetrachloro-3α-6α-diphenylglycouril
(IODO-Gen, Pierce) according to the manufacturer’s
instructions.
Gamma scintigraphy analysis
Biodistribution studies were performed in male New
Zealand White rabbits. Hypnorm sedated animals
(n = 4; average mass 1.0 kg) were injected intravenously
with 123I-labeled enzyme (2.5 mg/kg in < 1 ml; ~3 MBq)
solution in PBS. Imaging was done on a Maxi Gamma
Camera 406 (GE Medical Systems) at 1, 10, 30, 60, 90,
and 120 min.
Results and discussion
Design and synthesis of the prodrugs
We hypothesized that rhamnose-capped Dox 11 would
fragment according to the mechanism depicted in
Scheme 2 on enzymatic hydrolysis by α-rhamnosidase to
release Dox 9; loss of rhamnosyl would lead to carbamic
acid formation followed by spontaneous decarboxyla-
tion. An extended aminomethylcarbamate linker was
used for 5Fu prodrug 8 (Scheme 2) based on the study
by Monneret and co-workers for glucuronide-based
prodrugs (Madec-Lougerstay et al., 1999). e mode of
collapse of aminomethylcarbamate linker also involves
rhamnosyl loss followed by decarboxylation and aminal
hydrolysis.
e Dox prodrug 11 was also prepared from com-
pound 4 and doxorubicin hydrochloride (9) (Scheme 3).
e reaction of the doxorubicin amine group with acti-
vated rhamnose 4 proceeded readily in the presence of
1.5 eq. of pyridine to afford carbamate 10. Deacetylation
of compound 10 required precise conditions. When car-
ried out using 0.1 M sodium methoxide in dry MeOH
at 0°C, the reaction proceeded almost quantitatively
on a small scale (100 mg). However, during attempts to
scale up (to >1.5 g), the reaction failed and formation of
e same l-rhamnosyl precursor 4, containing a car-
bonate moiety as a precursor to carbamate formation,
was used for the synthesis of both 5Fu and Dox prod-
rugs, 8 and 11, respectively (Scheme 3). l-(+)-Rhamnose
© 2010 Informa UK, Ltd.

800 P. Garnier et al.
insoluble products was observed both during the reaction
and after quenching with acidic Dowex® resin. Inspired
by Florent et al. (1998), a mixture of MeOH/DMF/THF
(100/20/5) was used as a solvent instead of neat MeOH
(Scheme 3). In this new solvent system, no precipitation
was observed during the reaction or after quenching with
Dowex-H+ resin and filtration. Overall, this last step was
achieved in 94% yield, about twice the yield obtained in
neat MeOH.
e ASGPR, of which 50,000 to 500,000 copies
(Eisenberg et al., 1991) are typically present on the sur-
face of a hepatocyte cell, has been shown to provide an
efficient target for carbohydrate-mediated drug delivery
to the liver by endocytosis. Although individual carbo-
hydrate ligand to receptor interactions are typically not
strong enough to mediate efficient targeting, these can be
greatly enhanced by taking advantage of the multivalent
(or cluster) effect; this can be achieved by displaying mul-
tiple copies of a monosaccharide or branched saccharides
at the surface of a macromolecule. Impressive enhance-
ment of ASGPR binding of up to 1×106-fold have been
obtained for tetra-antennary Gal-terminated ligands over
monoantennary-Gal-terminated ones (Lee et al., 1983).
erefore, a strategy involving the attachment of multiple
copies of Gal monosaccharides was selected for the gly-
coengineering of N-WT to target it to the ASGPR. Protein
glycosylation can be carried out using a variety of chemi-
cal methods that offers key advantages such as versatility
(choice of carbohydrate structure), selectivity (choice of
glycosylation site), and ease of scale-up (Gamblin et al.,
2009). For this study, we chose to use the IME method
(Lee et al., 1976; Stowell and Lee, 1982) to modify lysine
residues on N-DG. e enzymes N-WT and N-DG were
incubated with monosaccharide Gal IME reagent to afford
N-WT-Gal and N-DG-Gal, respectively. ese synthetic
glycoproteins were characterized by gel electrophoresis
and mass spectrometry. Matrix-assisted laser desorption
ionization MS analysis showed an average incorporation
of 11–15 Gal residues per enzyme molecule: N-WT 76148;
Enzyme glycoengineering
Naringinase, produced by the fungus P. decumbens,
offers a ready source of α-l-rhamnosidase. Purified
N-WT was obtained in four steps from the dialyzed nar-
inginase. After dialysis into deionised water, the crude
naringinase solution was separated by a short Bio-Gel
P-100 column eluted with deionised water to remove
most of the small molecular weight impurities. A sec-
ond round of Bio-Gel P-100 chromatography separated
α-l-rhamnosidase from fractions containing β-d-
glucosidase. Finally, ion-exchange chromatography on
a DEAE Sepharose column separated α-l-rhamnosidase
from the remaining protein impurities. e yields (w/w)
were 24, 45, and 54% for three steps respectively, with
~6% overall yield.
Naringinase produced by P. decumbens carries a
mannose-rich motif (Richards and Leitch, 1989), which
were removed to avoid any undesired ligand-mediated
interaction. Treatment of N-WT with endo-β-N-
acetylglucosaminidase H (endo-H) yielded N-DG.
O
OH
O
O
OAc
OH
a
b
c
O
O
O
O
HO
AcO
AcO
AcO
NO2
HO
AcO
AcO
AcO
OH
OAc
OAc
OAc
4
1
2
3
O
O
O
O
F
F
N
N
O
N
COOH
O
N
H
OAc
O
N
H
O
N
H
H
d
e
f
g
O
O
O
O
O
N
H
O
O
N
H
O
AcO
AcO
AcO
HO
4
AcO
AcO
AcO
HO
OAc
OAc
OAc
OH
8
7
5
6
O
O
OH
OH
O
O
OH
OH
O
OH
OH
OH
OH
O
O
OH
O
OH
CH3O
AcO
O
CH3O
O
O
OH
h
i
O
O
CH3O
OH O
NH
HO
NH
HO
O
O
O
O
O
NH2
HO
O
O
HO
9
AcO
HO
OAc
10
OH
11
Reaction conditions: a) Ac2O, pyridine, rt, quant.; b) BnNH2, THF, 91 %; c) 4-nitrophenyl chloroformate, pyridine (1 eq.), DCM, 60 %;
d) glycine, NaHCO3, water/acetone (7/3), 80 %; e) Pb(OAc)4, pyridine, THF/toluene, 35 %; f) 5-fluorouracil, Et3N, DMF, 77 %; g)
0.1 M NaOMe, MeOH, quant.; h) 4, Et3N (1.5 eq.), DMF, 94 %; i) 0.016 M NaOMe, MeOH/DMF/THF (50/10/2.5), 94 %.
Scheme 3. Synthesis of 5-fluorouracil and doxorubicin prodrugs.
Journal of Drug Targeting

Synthesis and evaluation of rhamnose-capped prodrugs 801
N-WT-Gal 79443 (N-WT + 3295 ~+14 Gal); N-DG 69341;
N-DG-Gal 71892 (NDG + 2551 ~+11 Gal).
(Smedsrød et al., 1994). Male New Zealand white rab-
bits were dosed with N-DG-Gal, N-DG-Gal + Rha-Dox,
or N-DG-Gal + Rha-5Fu. Images were acquired using a
Maxi Gamma Camera 406 (GE Medical Systems, Slough,
UK) at 1, 10, 30, 60, 90, and 120 min after administration.
Analysis was carried out using a MICAS X image process-
ing system (Bartec Technologies, Camberley, Surrey, UK).
As can be seen on the time course distribution shown in
Figure 1, strong localization of the 123I-labeled N-DG-Gal
was achieved in the liver, with a smaller amount detected
in the kidney and bladder area. Importantly, coadmin-
istration of the rhamnose-capped prodrugs Rha-Dox or
Rha-5Fu did not significantly affect enzyme localization.
ese observations are consistent with results obtained in
our previous study for N-DG-Gal alone (Robinson et al.,
2004), which had established that liver uptake is strongly
dependent on its sugar coat. Taken together, these results
suggest that dosing of the prodrugs together with N-DG-
Gal does not lead to inhibition of N-DG-Gal uptake.
Blood stability and cap-release studies
Rha-Dox 11 was incubated at three different concen-
trations for 60 min in either 0.9% NaCl or rabbit blood
to assess its stability as determined by HPLC analysis
(Table 2). Although Rha-Dox 11 was, as expected, stable
in 0.9% NaCl, a significant decrease was observed for
blood samples of Rha-Dox after incubation at 37°C.
However, no significant amount of Dox 9 was detected
by HPLC, which suggests that the lower levels of Rha-
Dox observed in blood cannot be attributed to hydroly-
sis of Rha-Dox by glycosidases present in blood. Other
mechanisms, for example, binding with blood proteins
or degradation by other pathways or absorption at the
surface of blood cells, could account for the drop in Rha-
Dox titres that were observed.
e Rha-5Fu prodrug 8 was shown to be very stable in
both 0.9% NaCl and in blood, as shown in Table 3. Almost
quantitative recovery of the intact prodrug was observed
after 60 min incubation in rabbit blood.
Conclusions
Incubation of Rha-Dox 11 and Rha-5Fu 8 in both
0.9%NaCl and blood with both N-WT-Gal and N-DG-Gal
showed ready release of Dox and 5Fu, respectively. is
importantly confirmed both the activity of synthetically
glycosylated enzymes and the liability of the prodrugs to
these enzymes.
Two rhamnose-capped prodrugs of the common cyto-
toxic drug Dox and 5Fu have been synthesized. eir
demonstrated stability in blood makes them suitable for
the LEAPT strategy as they do not undergo unwanted
hydrolysis before reaching their target, in this case the
hepatocytes. ese findings complement the previously
demonstrated enhancement in stability shown for the
glycosylated enzyme N-DG-Gal (Robinson et al., 2004);
chemical galactosylation of N-DG to form N-DG-Gal
resulted in increased enzyme stability, extending its half-
Effect of prodrugs on time course of distribution of
synthetically glycosylated enzymes using gamma
scintigraphy
Determination of the in vivo distribution of macromol-
ecules can be achieved in a noninvasive manner by using
gamma scintigraphy. Spatial distribution to the different
organs can be determined quantitatively with 123I labeling
10min
30min
60min
120min
Table 2. Stability of Rha-Dox after 60min in 0.9% NaCl or rabbit
blood.
N-DG-Gal
Initial Rha-Dox
concentration (μg/ml)
% Recovery
(deviation)
0.9% NaCl
100
10
1
100.6 (1.8)
100.5 (1.0)
101.8 (3.4)
62.3 (1.7)
65.6 (1.2)
55.1 (1.2)
Rabbit blood
100
10
1
Rha-Dox + N-DG-Gal
Table 3. Stability of Rha-5Fu after 60min in 0.9% NaCl or rabbit
blood.
Initial Rha-5Fu
concentration (μg/ml)
Rha-5Fu + N-DG-Gal
% Recovery
99.9
0.9% NaCl
200
20
2
99.8
101.5
97.2
Rabbit blood
200
20
2
96.5
Figure 1. Gamma scintigraphy time-course determination of in
vivo organ distribution.
94.6
© 2010 Informa UK, Ltd.

802 P. Garnier et al.
life from 1h for N-DG to more than 48h for N-DG-Gal.
Such results, suggesting increased stability of synthetically
glycosylated enzymes, tally with those recently deter-
mined against proteolytic enzymes (Russell et al., 2009).
Rhamnosidase N-WT was glycoengineered to retune its
binding specificity by alteration of its natural glycosylation
pattern (to form N-DG) and subsequent modification by
the attachment of multiple Gal moieties (to form N-DG-
Gal) to specifically target the ASGPR and simultaneously
take advantage of a multivalent effect to enhance binding.
Time-course biodistribution studies described here show
very clearly that N-DG-Gal is very quickly taken-up by the
liver, and a smaller portion goes to the kidney and blad-
der. ese results are consistent with the results obtained
in a previous study, in which the in vivo distribution of the
enzyme was assessed for each organ (liver, kidneys, blad-
der, heart/lung, brain by gamma scintigraphy) and showed
rapid uptake for N-DG-Gal and rapid serum clearance
(Robinson et al., 2004). Importantly, coadministration of
prodrug (Rha-Dox or Rha-5Fu) does not inhibit N-DG-Gal
uptake allowing uninterfered colocalization of both key
components of the LEAPT system.
All the results presented here analyzing the synthesis
and behavior of rhamnosyl prodrugs in the LEAPT system
are consistent with results previously reported (Robinson
et al., 2004) in which Rha-Dox prodrug was successfully
evaluated in a disease model study of the LEAPT system.
is prior study showed promising approximate halv-
ing in tumor burden and foci after 42 days of three-time
weekly dosing compared with control groups of prodrug
or N-DG-Gal alone. e efficacy of other prodrugs, such
as Rha-5Fu, shown here for delivery of, for example, 5Fu
suggests the modularity of the LEAPT approach. In prin-
ciple, while using different drugs in this system, any suit-
able prodrug cap may also be employed (allowing use of
many enzymes) and any sugar type may be used to deco-
rate the releasing enzyme (allowing targeting to a wide
range of cognate endogenous sugar-binding lectins).
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Declaration
B.G.D. and A.J.F. are shareholders in Glycoform, PG holds
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